Development And Application Of AFB₁,T-2 And DON Immunochromatographic Strip

Mycotoxin is a toxic metabolite produced by fungus which can contaminate feed and food and cause animals poisoning.The contamination of mycotoxins in China is very serious.Every year,about 20%of the feed is discarded due to mycotoxin contamination,resulting in severe economic losses.Among the identified fungal toxins,the major ones that are more harmful include:aflatoxin toxin(AFT),deoxynivalenol(DON),T-2 toxin(T-2),zearalenone(ZEA),etc.Grains grown in nature are vulnerable to the contamination of a variety of mycotoxins.When two or more mycotoxins co-exist,they may produce a combination or antagonism effect,but the combination effect is more common.Mycotoxin poisoning poses a serious threat to human and animal health,so it is especially important to establish a convenient and effective detection method of mycotoxins.Common methods of detection and analysis include:thin layer chromatography(TLC),high performance liquid chromatography-mass spectrometry(HPLC-MS),and enzyme linked immunosorbent assay(ELISA).The advantages of TLC are simple operation method and low cost,but the workload of processing samples is large,and the operators have to contact the standard products directly,which will endanger the health of the operators.The detection instrument of HPLC is expensive,and the sample pretreatment is more complex.Besides,the operation requires special technicians,so that it's not suitable for the detection of large quantities of feed.ELISA is fast and sensitive,but it needs to be washed many times.And the detection time is too long to meet the requirements of field detection.Therefore,in order to ensure the health of humans and animals,it is important to establish a rapid screening and detection method of mycotoxins.Aflatoxin B1(AFB1),T-2 Toxin(T-2)and deoxynivalenol(DON),which is known as the highly toxic and widely distributed mycotoxins,are selected as the research objects in this study.The method of rapid detection of AFB1,DON,and T-2 was established by immunological principle.The main research contents are as follows:1.Preparation of AFB1 monoclonal antibodiesAFB1-BSA was used as complete antigen to immunize BALB/c mice to obtain high specific serum.Through cell fusion and subcloning,a total of 10 cell cell strains were obtained to produce aflatoxin B1 specific monoclonal antibodies.1C10 cell lines were selected to produce monoclonal antibodies and monoclonal antibodies by ascites,and then the subtype,affinity and cross reaction rate of the antibodies were identified.The concentration of AFB1 monoclonal antibody was 0.8 mg·L-1.The subtype of the IgG antibody is IgG1.2.Development of the AFB1,DON and T-2 immunochromatographic stripsOn the basis of the available monoclonal antibodies of AFB1,DON and T-2,we developed a colloidal gold test strip which can detect three kinds of mycotoxins simultaneously.And it is highly sensitive and suitable for field testing.It mainly consist of preparation of colloidal gold,identification of gold particles,determination of the dosage of colloidal gold antibody,optimization of the coating concentration of the antigen on the detection line,the assembly of multiple test strip,etc.When the detection line disappeared,the concentration of the minimum mycotoxin in the feed sample diluent were 2 ng·mL-1 of AFB1,20 ng·mL-1 of T-2 and 100 ng·mL-1 of DON,respectively.That is to say,the detection line would disappear if the concentration of mycotoxins in 1g feed reached 20 ng·kg-1 of AFB1,200 ng·kg-1 of T-2 and 1000 ng·kg-1 of DON respectively,which indicates that the mycotoxin content of the sample exceeds the standard.3.Colloidal gold paper strip test for actual sample.The specificity,sensitivity,repeatability and stability of the trigeminal colloidal gold paper strip were detected.If the detection result was corresponded to HPLC detected,the strip could be used to detect the related mycotoxin.The colloidal gold paper strip were used to test 186 feeds that were obtained from the feed mills of Shandong province and Henan province.The results showed that the contamination rate of AFB1 was 83%,relatively serious,and that of DON was 56%,and that of T-2 was 20%.